Kallikrein-Inhibitor Therapies

ABSTRACT

Methods are described for preventing or reducing ischemia, e.g., cerebral ischemia, and/or reperfusion injury, e.g., reperfusion injury associated with cerebral ischemia, in a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/466,979, filed Aug. 24, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/953,902, filed on Sep. 27, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/456,986,filed Jun. 6, 2003, which claims priority to U.S. provisionalapplication No. 60/387,239, filed on Jun. 7, 2002, and 60/407,003, filedon Aug. 28, 2002, the contents of all of which are incorporated hereinby reference.

SUMMARY

Serine proteases such as, kallikrein (e.g., plasma kallikrein), areinvolved in pathways leading to excessive perioperative blood loss, theonset of systemic inflammatory response and nervous systempathophysiology. Inhibitors of kallikrein include proteinaceous andnon-proteinaceous molecules. Exemplary plasma kallikrein inhibitorsinclude those described in U.S. Pat. Nos. 6,333,402 and 6,057,287, thecontents of which are incorporated herein by reference in theirentirety.

Polypeptide and other inhibitors of plasma kallikrein can be used intherapeutic methods and compositions suitable for use in eliminating orreducing various ischemias, including but not limited to perioperativeblood loss, cerebral ischemia, the onset of systemic inflammatoryresponse, and/or reperfusion injury, e.g., reperfusion injury associatedwith cerebral ischemia or a focal brain ischemia. Perioperative bloodloss results from invasive surgical procedures that lead to contactactivation of complement components and the coagulation/fibrinolysissystems. The kallikrein inhibitors can be used to reduce or preventperioperative blood loss and a systemic inflammatory response inpatients subjected to invasive surgical procedures, especiallycardiothoracic surgeries. The kallikrein inhibitors can also be used toreduce or prevent cerebral ischemia such as stroke, and/or reperfusioninjury associated with cerebral ischemia. They can also preventneurological and cognitive deficits associated with stroke, blood lose,and cerebral ischemia, e.g., events that are not associated withsurgical intervention. The inhibitors can be administered prior to,during, or after an event, e.g., a cardiovascular event that can damagethe central nervous system.

A variety of inhibitors of a kallikrein, e.g., a plasma kallikrein aredescribed herein. Any one of these, or a combination of more than one ofthese, can be used in the following methods.

In one aspect, the disclosure features a method for preventing orreducing ischemia in a patient including administering to the patient acomposition that includes an inhibitor of kallikrein, e.g., a plasmakallikrein. Typically the patient is a human patient. The compositioncan be administered in an amount effective to prevent or reduce ischemiain the patient. In a particular embodiment, the ischemia is at leastpartially due to blood loss, e.g., perioperative blood loss due to asurgical procedure performed on the patient. The surgical procedure canbe, e.g., a cardiothoracic surgery, such as, for example,cardiopulmonary bypass or coronary artery bypass grafting. The inhibitorcan be administered before, during, or after the procedure.

In another aspect, the disclosure features a method for preventing orreducing a systemic inflammatory response, e.g., a response associatedwith a surgical procedure in a patient or its onset. The methodincludes: administering to the patient a composition including aninhibitor of kallikrein, e.g., a plasma kallikrein. The composition canbe administered before, during, or after surgery. In one embodiment, thesurgical procedure is a cardiothoracic surgery, such as, for example,cardiopulmonary bypass or coronary artery bypass grafting.

In another aspect, the disclosure features a method for treating a brainor central nervous system (CNS) injury. The method can be used toprevent or reduce adverse effects of cerebral ischemia, e.g., stroke,and/or reperfusion injury, e.g., reperfusion injury associated withcerebral ischemia, in a patient including administering to the patient acomposition including an inhibitor of kallikrein, e.g., a plasmakallikrein. In one embodiment, the cerebral ischemia is stroke, e.g.,embolism-, thrombus- or hemorrhage-associated stroke. The method caninclude administering the inhibitor, before, during, or after theischemia, e.g., at the time of reperfusion or at a time between 1-12hours after an ischemic event, e.g., between 1-5 hours after such anevent.

The inhibitor used in any disclosed method can have a Ki for kallikrein,e.g., plasma kallikrein of less than 50 nM, 5 nM, 1 nM, 500 pM, 100 pM,50 pM, e.g., about 44 pM. The inhibitor can preferentially inhibitplasma kallikrein at least 100, 200, 500, or 1000 more than anotherkallikrein, e.g., human urine kallikrein, or another protease, e.g.,plasmin or thrombin. For example, the inhibitor is other than aprotinin.

In one embodiment, the inhibitor is an agent that can cross theblood-brain barrier.

In one embodiment, the inhibitor includes a polypeptide that includes aKunitz domain such as the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4 CysXaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 CysXaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 CysXaa56 Xaa57 Xaa58 (SEQ ID NO:1).

The framework of the Kunitz domain can be human or can differ from ahuman Kunitz domain framework by fewer than six, five, four, three, ortwo amino acids. For example, the framework of the Kunitz domain can bethe framework of one of the Kunitz domains of humanlipoprotein-associated coagulation inhibitor (LACI) protein, e.g., thefirst second or third Kunitz domain. Typically the polypeptide differsfrom BPTI and/or one or more of the LACI Kunitz domains by at least one,two, three, or four amino acids, e.g., at least one, two or three aminoacids in the binding loops and/or at least two, three, four, or sixamino acids in the framework region. For example, the polypeptide caninclude a non-naturally occurring Kunitz domain that is derived from anaturally occurring Kunitz domain, e.g., a human Kunitz domain. In oneembodiment, an inhibitor that includes a Kunitz domain binds to plasmakallikrein with an affinity that is at least 10, 100, or 500 fold betterthan BPTI and/or LACI.

In one embodiment, the polypeptide that inhibits kallikrein is notimmunogenic on second use.

In one embodiment, the polypeptide can have one or more of the followingfeatures: Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are eachindividually an amino acid or absent; Xaa10 is an amino acid selectedfrom the group consisting of: Asp and Glu; Xaa11 is an amino acidselected from the group consisting of: Asp, Gly, Ser, Val, Asn, Ile, Alaand Thr; Xaa13 is an amino acid selected from the group consisting of:Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an aminoacid selected from the group consisting of: Arg, Lys, Ala, Ser, Gly,Met, Asn and Gln; Xaa16 is an amino acid selected from the groupconsisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an amino acidselected from the group consisting of: Ala, Asn, Ser, Ile, Gly, Val, Glnand Thr; Xaa18 is an amino acid selected from the group consisting of:His, Leu, Gln and Ala; Xaa19 is an amino acid selected from the groupconsisting of: Pro, Gln, Leu, Asn and Ile; Xaa21 is an amino acidselected from the group consisting of: Trp, Phe, Tyr, His and Ile; Xaa22is an amino acid selected from the group consisting of: Tyr and Phe;Xaa23 is an amino acid selected from the group consisting of: Tyr andPhe; Xaa31 is an amino acid selected from the group consisting of: Glu,Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acidselected from the group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu,Ser, Ala, Gly and Val; Xaa34 is an amino acid selected from the groupconsisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is anamino acid selected from the group consisting of: Tyr, Trp and Phe;Xaa39 is an amino acid selected from the group consisting of: Glu, Gly,Ala, Ser and Asp; Xaa40 is an amino acid selected from the groupconsisting of: Gly and Ala; Xaa43 is an amino acid selected from thegroup consisting of: Asn and Gly; Xaa45 is an amino acid selected fromthe group consisting of: Phe and Tyr; and wherein the polypeptideinhibits kallikrein.

In a particular embodiment, individual amino acid positions of SEQ IDNO:1 can be one or more of the following: Xaa10 is Asp, Xaa11 is Asp,Xaa13 is Pro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His,Xaa19 is Pro, Xaa21 is Trp, Xaa31 is Glu, Xaa32 is Glu, Xaa34 is Ile,Xaa35 is Tyr, Xaa39 is Glu.

The polypeptide can include (or consist of) the following amino acidsequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly ProCys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu GluGlu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2), or a fragmentthereof, e.g., amino acids 3-60 of SEQ ID NO:2 or other fragment thatbinds and inhibits kallikrein. For example, the polypeptide can havefewer than 80, 70, 65, or 60 amino acids.

In one embodiment, the polypeptide is modified, e.g., to include one ormore polymer moieties, e.g., a plurality of polymer moieties, e.g., asdescribed in U.S. Ser. No. 10/931,153, filed Aug. 30, 2004, bearingattorney docket number 10280-119001. For example, the polypeptide caninclude a plurality of polyethylene glycol moieties, e.g., one on anN-terminal amine and one attached to each lysine of the polypeptide. Thepolyethylene glycol moieties can be less than 10, 8, 7, or 6 kDa inaverage molecular weight. Other exemplary modifications include a label,e.g., a radioactive or MRI-detectable label.

In one embodiment, the inhibitor does not include a peptide orpolypeptide. For example, the inhibitor can be small molecule, e.g., acompound described in WO 04/062657, e.g., an acylated 4-amidino- or4-guanidinobenzylamines. The compound can have the general formula (I)P4-P3-P2-P1 (I), where P4 is a mono- or poly-substituted orunsubstituted benzylsulphonyl group, P3 is a mono- or poly-substitutedor unsubstituted, natural or unnatural alpha-amino or alpha-imino acidwith the D-configuration, P2 is a mono- or poly-substituted orunsubstituted natural or unnatural alpha-amino or alpha-imino acid withthe L-configuration and P1 is a mono- or poly-substituted orunsubstituted 4-amidino- or 4-guanidinobenzylamine group.

The methods described herein can include administering an effectiveamount of the inhibitor of kallikrein. Such an amount can be an amountsufficient to produce an improvement detectable to one skilled in theart, to ameliorate at least one symptom, or to modulate (e.g., improve)at least one physiological parameter, e.g., to a statisticallysignificant degree.

All patents, patent applications, and publications cited herein areincorporated herein by reference. In the case of conflict, the presentapplication controls.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified diagram of major multiple pathways and relatedevents involved in the contact activation system and systemicinflammatory response (SIR) that may arise in a patient subjected tosoft and bone tissue trauma such as that associated with a coronaryartery bypass grafting (CABG) procedure, especially when the CABGprocedure involves extra-corporeal blood circulation, such ascardiopulmonary bypass (Bypass Apparatus). Arrows indicate activationfrom one component or event to another component or event in thecascade. Arrows in both directions indicate activating effects ofcomponents or events in both directions. Broken arrows indicate likelyparticipation of one component or event in the activation of anothercomponent or event. Abbreviations: “tPA”, tissue plasminogen activator;“C5a”, a protein component of the complement system; “fXIIa”, activatorprotein of prekallikrein to form active kallikrein; “Extrinsic”,extrinsic coagulation system; “Intrinsic”, intrinsic coagulation system.

FIG. 2 shows a portion of a DNA and corresponding deduced amino acid foran exemplary kallikrein inhibitor polypeptide in plasmid pPIC-K503. Theinserted DNA encodes the matα Prepro signal peptide of Saccharomycescerevisiae (underlined) fused in frame to the amino terminus of thePEP-1 polypeptide having the amino acid sequence enclosed by the boxedarea. The amino acid sequence of the PEP-1 polypeptide shown in theboxed region is SEQ ID NO:2, and the corresponding nucleotide codingsequence is SEQ ID NO:3. The dashed arrows indicate the location anddirection of two PCR primer sequences in AOX regions that were used toproduce sequencing templates. DNA sequence for the entire nucleotidesequence of the figure includes the structural coding sequence for thefusion protein and is designated SEQ ID NO:27. The double underlinedportion of the sequence indicates a diagnostic probe sequence. BstB Iand EcoR I indicate locations of their respective palindromic,hexameric, restriction endonuclease sites in the sequence. Asterisksdenote translational stop codons. See text for details.

FIGS. 3A and 3B show an alignment of exemplary amino acid sequences, thenative LACI sequence from which these variants were derived (SEQ IDNO:32), and other known Kunitz domains (SEQ ID NOS:29-31 and 33-53).Cysteine residues are highlighted.

FIG. 4 is a graph depicting ischemic volume in C57B1/6 mice subjected totransient focal cerebral ischemia by middle cerebral artery occlusion(MCAO) using 5-0 microfilament and administered saline (control) or akallikrein inhibitor polypeptide (DX-88) at various doses (10 Tg, 30 Tg,90 Tg). The saline or kallikrein inhibitor polypeptide was administeredat the beginning of the ischemic period.

FIG. 5 is a graph depicting ischemic volume in C57B1/6 mice subjected totransient focal cerebral ischemia by middle cerebral artery occlusion(MCAO) using 6-0 microfilament and administered saline (control) or akallikrein inhibitor polypeptide (DX-88) at various periods before andafter ischemic damage. The kallikrein inhibitor polypeptide wasadministered at the beginning of the ischemic period (DX-88 pre), at theend of the ischemic period (DX-88 post), or at one hour after thebeginning of the ischemic period (DX-88 1 h post). The kallikreininhibitor polypeptide was administered at 30 Tg.

FIG. 6 is a graph depicting general neurological deficits in C57B1/6mice subjected to transient focal cerebral ischemia by middle cerebralartery occlusion (MCAO) using 6-0 microfilament and administered saline(control) or a kallikrein inhibitor polypeptide (DX-88) at variousperiods before and after ischemic damage. The kallikrein inhibitorpolypeptide was administered at the beginning of the ischemic period(DX-88 pre), at the end of the ischemic period (DX-88 post), or at onehour after the beginning of the ischemic period (DX-88 1 h post). Thekallikrein inhibitor polypeptide was administered at 30 Tg. Generalneurological deficits were measured based upon evaluation of the hair,ears, eyes, posture, spontaneous activity and eptileptic behavior in themice.

FIG. 7 is a graph depicting focal neurological deficits in C57B1/6 micesubjected to transient focal cerebral ischemia by middle cerebral arteryocclusion (MCAO) using 6-0 microfilament and administered saline(control) or a kallikrein inhibitor polypeptide (DX-88) at variousperiods before and after ischemic damage. The kallikrein inhibitorpolypeptide was administered at the beginning of the ischemic period(DX-88 pre), at the end of the ischemic period (DX-88 post), or at onehour after the beginning of the ischemic period (DX-88 1 h post). Thekallikrein inhibitor polypeptide was administered at 30 Tg. Focalneurological deficits were measured based upon evaluation of the bodysymmetry, gait, climbing ability, circling behavior, front limbsymmetry, compulsory circling and whisker response in the mice.

FIG. 8 is a graph depicting the levels of a kallikrein activity found inthe cerebral spinal fluid (CSF) of either untreated mice (normal CSF) ormice treated with a kallikrein inhibitor polypeptide (DX-88) at variousdoses (10 Tg or 30 Tg) measured at 30 or 60 minutes afteradministration.

DETAILED DESCRIPTION

Kallikrein inhibitors, particularly inhibitors of plasma kallikrein, canbe used to prevent or treat disorders associated with blood loss orblood fluidity. For example, such inhibitors can be used to treat orprevent perioperative blood loss, a systemic inflammatory response (SIR)induced by kallikrein (especially, for example, in patients undergoingsurgical procedures and particularly surgical procedures involvingcardiothoracic surgery, e.g., cardiopulmonary bypass (CPB), such as acoronary artery bypass graft (CABG) procedures as well as in patientswith other disorders), cerebral ischemia and/or reperfusion injuryassociated with ischemia, e.g., cerebral ischemia.

Further examples of applications for kallikrein inhibitors includepediatric cardiac surgery, lung transplantation, total hip replacementand orthotopic liver transplantation, and to reduce or preventperioperative stroke during CABG, extracorporeal membrane oxygenation(ECMO) and cerebrovascular accidents (CVA) during these procedures.Kallikrein inhibitors can also be used for stroke, e.g., embolism,thrombus and/or hemorrhage associated stroke and for reperfusion injuryassociated with stroke.

Cardiothoracic surgery is surgery of the chest area, most commonly theheart and lungs. Typical diseases treated by cardiothoracic surgeryinclude coronary artery disease, tumors and cancers of the lung,esophagus and chest wall, heart vessel and valve abnormalities, andbirth defects involving the chest or heart. Where cardiothoracic surgeryis utilized for treatment, the risk of blood loss (e.g., surgery-inducedischemia) and the onset of a systemic inflammatory response (SIR) isincurred. Surgery-induced SIR can result in severe organ dysfunction(systemic inflammatory response syndrome; SIRS).

Kunitz Domains

A number of useful inhibitors of kallikrein include a Kunitz domain.

As used herein, a “Kunitz domain” is a polypeptide domain having atleast 51 amino acids and containing at least two, and preferably three,disulfides. The domain is folded such that the first and sixthcysteines, the second and fourth, and the third and fifth cysteines formdisulfide bonds (e.g., in a Kunitz domain having 58 amino acids,cysteines can be present at positions corresponding to amino acids 5,14, 30, 38, 51, and 55, according to the number of the BPTI sequenceprovided below, and disulfides can form between the cysteines atposition 5 and 55, 14 and 38, and 30 and 51), or, if two disulfides arepresent, they can form between a corresponding subset of cysteinesthereof. The spacing between respective cysteines can be within 7, 5, 4,3 or 2 amino acids of the following spacing between positionscorresponding to: 5 to 55, 14 to 38, and 30 to 51, according to thenumbering of the BPTI sequence provided below. The BPTI sequence can beused a reference to refer to specific positions in any generic Kunitzdomain. Comparison of a Kunitz domain of interest to BPTI can beperformed by identifying the best fit alignment in which the number ofaligned cysteines in maximized.

The 3D structure (at high resolution) of the Kunitz domain of BPTI isknown. One of the X-ray structures is deposited in the BrookhavenProtein Data Bank as “6PTI”. The 3D structure of some BPTI homologues(Eigenbrot et al., (1990) Protein Engineering, 3(7):591-598; Hynes etal., (1990) Biochemistry, 29:10018-10022) are known. At least seventyKunitz domain sequences are known. Known human homologues include threeKunitz domains of LACI (Wun et al., (1988) J. Biol. Chem.263(13):6001-6004; Girard et al., (1989) Nature, 338:518-20; Novotny etal, (1989) J. Biol. Chem., 264(31):18832-18837) two Kunitz domains ofInter-α-Trypsin Inhibitor, APP-I (Kido et al., (1988) J. Biol. Chem.,263(34):18104-18107), a Kunitz domain from collagen, and three Kunitzdomains of TFPI-2 (Sprecher et al., (1994) PNAS USA, 91:3353-3357). LACIis a human serum phosphoglycoprotein with a molecular weight of 39 kDa(amino acid sequence in Table 1) containing three Kunitz domains.

TABLE 1 Exemplary Natural Kunitz Domains LACI: (SEQ ID NO. 54)   1MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit dtelpplklM  51 HSFCAFKADDGPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC 101 KKMCTRDnan riikttlqqekpdfCfleed pgiCrgyitr yfynnqtkqC 151 erfkyggClg nmnnfetlee CkniCedgpngfqvdnygtq lnavnnsltp 201 qstkvpslfefhgpswCltp adrglCrane nrfyynsvig kCrpfkysgC 251 ggnennftsk qeClraCkkgfiqriskggl iktkrkrkkq rvkiayeeif 301 vknm The signal sequence (128) isuppercase and underscored LACI-K1 is uppercase LACI-K2 is underscoredLACI-K3 is bold BPTI     1    2    3    4    5 (SEQ ID NO:55)1234567890123456789012345678901234567890123456789012345678RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA

The Kunitz domains above are referred to as LACI-K1 (residues 50 to107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to 270). The cDNAsequence of LACI is reported in Wun et al. (J. Biol. Chem., 1988,263(13):6001-6004). Girard et al. (Nature, 1989, 338:518-20) reportsmutational studies in which the P1 residues of each of the three Kunitzdomains were altered. LACI-K1 inhibits Factor VIIa (F.VIIa) when F.VIIais complexed to tissue factor and LACI-K2 inhibits Factor Xa.

Proteins containing exemplary Kunitz domains include the following, withSWISS-PROT Accession Numbers in parentheses:

A4_HUMAN (P05067), A4_MACFA (P53601), A4_MACMU (P29216), A4_MOUSE(P12023), A4_RAT (P08592), A4_SAISC (Q95241), AMBP_PLEPL (P36992),APP2_HUMAN (Q06481), APP2_RAT (P15943), AXP1_ANTAF (P81547), AXP2_ANTAF(P81548), BPT1_BOVIN (P00974), BPT2_BOVIN (P04815), CA17_HUMAN (Q02388),CA36_CHICK (P15989), CA36_HUMAN (P12111), CRPT_BOOMI (P81162),ELAC_MACEU (O62845), ELAC_TRIVU (Q29143), EPPI_HUMAN (O95925),EPPI_MOUSE (Q9DA01), HT1B_MANSE (P26227), IBP_CARCR (P00993), IBPC_BOVIN(P00976), IBPI_TACTR (P16044), IBPS_BOVIN (P00975), ICS3_BOMMO (P07481),IMAP_DROFU (P11424), IP52_ANESU (P10280), ISC1_BOMMO (P10831),ISC2_BOMMO (P10832), ISH1_STOHE (P31713), ISH2_STOHE (P81129),ISIK_HELPO (P00994), ISP2_GALME (P81906), IVB1_BUNFA (P25660),IVB1_BUNMU (P00987), IVB1_VIPAA (P00991), IVB2_BUNMU (P00989),IVB2_DABRU (P00990), IVB2_HEMHA (P00985), IVB2_NAJNI (P00986), IVB3VIPAA (P00992), IVBB_DENPO (P00983), IVBC_NAJNA (P19859), IVBC_OPHHA(P82966), IVBE_DENPO (P00984), IVBI_DENAN (P00980), IVBI_DENPO (P00979),IVBK_DENAN (P00982), IVBK_DENPO (P00981), IVBT_ERIMA (P24541),IVBT_NAJNA (P20229), MCPI_MELCP (P82968), SBPI_SARBU (P26228),SPT3_HUMAN (P49223), TKD1_BOVIN (Q28201), TKD1_SHEEP (Q29428),TXCA_DENAN (P81658), UPTI_PIG (Q29100), AMBP_BOVIN (P00978), AMBP_HUMAN(P02760), AMBP_MERUN (Q62577), AMBP_MESAU (Q60559), AMBP_MOUSE (Q07456),AMBP_PIG (P04366), AMBP_RAT (Q64240), IATR_HORSE (P04365), IATR_SHEEP(P13371), SPT1_HUMAN (O43278), SPT1_MOUSE (Q9R097), SPT2_HUMAN (O43291),SPT2_MOUSE (Q9WU03), TFP2_HUMAN (P48307), TFP2_MOUSE (O35536),TFPI_HUMAN (P10646), TFPI_MACMU (Q28864), TFPI_MOUSE (O54819),TFPI_RABIT (P19761), TFPI_RAT (Q02445), YN81_CAEEL (Q03610)

A variety of methods can be used to identify a Kunitz domain from asequence database. For example, a known amino acid sequence of a Kunitzdomain, a consensus sequence, or a motif (e.g., the ProSite Motif) canbe searched against the GenBank sequence databases (National Center forBiotechnology Information, National Institutes of Health, Bethesda Md.),e.g., using BLAST; against Pfam database of HMMs (Hidden Markov Models)(e.g., using default parameters for Pfam searching; against the SMARTdatabase; or against the Propom database. For example, the PfamAccession Number PF00014 of Pfam Release 9 provides numerous Kunitzdomains and an HMM for identify Kunitz domains. A description of thePfam database can be found in Sonhammer et al. (1997) Proteins28(3):405-420 and a detailed description of HMMs can be found, forexample, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskovet al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) ProteinSci. 2:305-314. The SMART database (Simple Modular Architecture ResearchTool, EMBL, Heidelberg, DE) of HMMs as described in Schultz et al.(1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (2000)Nucl. Acids Res 28:231. The SMART database contains domains identifiedby profiling with the hidden Markov models of the HMMer2 search program(R. Durbin et al. (1998) Biological sequence analysis: probabilisticmodels of proteins and nucleic acids. Cambridge University Press). Thedatabase also is annotated and monitored. The Propom protein domaindatabase consists of an automatic compilation of homologous domains(Corpet et al. (1999), Nucl. Acids Res. 27:263-267). Current versions ofPropom are built using recursive PSI-BLAST searches (Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computersand Chemistry 23:333-340.) of the SWISS-PROT 38 and TREMBL proteindatabases. The database automatically generates a consensus sequence foreach domain. Prosite lists the Kunitz domain as a motif and identifiesproteins that include a Kunitz domain. See, e.g., Falquet et al. NucleicAcids Res. 30:235-238 (2002).

Kunitz domains interact with target protease using, primarily, aminoacids in two loop regions (“binding loops”). The first loop region isbetween about residues corresponding to amino acids 15-20 of BPTI. Thesecond loop region is between about residues corresponding to aminoacids 31-37 of BPTI. An exemplary library of Kunitz domains varies oneor more amino acid positions in the first and/or second loop regions.Particularly useful positions to vary, when screening for Kunitz domainsthat interact with kallikrein or when selecting for improved affinityvariants, include: positions 13, 16, 17, 18, 19, 31, 32, 34, and 39 withrespect to the sequence of BPTI. At least some of these positions areexpected to be in close contact with the target protease. It is alsouseful to vary other positions, e.g., positions that are adjacent to theaforementioned positions in the three-dimensional structure.

The “framework region” of a Kunitz domain is defined as those residuesthat are a part of the Kunitz domain, but specifically excludingresidues in the first and second binding loops regions, i.e., aboutresidues corresponding to amino acids 15-20 of BPTI and 31-37 of BPTI.Conversely, residues that are not in the binding loop may tolerate awider range of amino acid substitution (e.g., conservative and/ornon-conservative substitutions).

In one embodiment these Kunitz domains are variant forms of the loopedstructure including Kunitz domain 1 of human lipoprotein-associatedcoagulation inhibitor (LACI) protein. LACI contains three internal,well-defined, peptide loop structures that are paradigm Kunitz domains(Girard, T. et al., 1989. Nature, 338:518-520). Variants of Kunitzdomain 1 of LACI described herein have been screened, isolated and bindkallikrein with enhanced affinity and specificity (see, for example,U.S. Pat. Nos. 5,795,865 and 6,057,287, incorporated herein byreference). These methods can also be applied to other Kunitz domainframeworks to obtain other Kunitz domains that interact with kallikrein,e.g., plasma kallikrein. Useful modulators of kallikrein functiontypically bind and/or inhibit kallikrein, as determined using kallikreinbinding and inhibition assays.

An exemplary polypeptide that includes a Kunitz domain that inhibitskallikrein has the amino acid sequence defined by amino acids 3-60 ofSEQ ID NO:2.

An exemplary polypeptide includes the amino acid sequence:

(SEQ ID NO:1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 GlyGly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58

“Xaa” refers to a position in a peptide chain that can be any of anumber of different amino acids. In a first example, Xaa can by anyamino acid except cysteine. In another example, one or more of thefollowing apply: Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser,Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr,Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn orGln; Xaa16 can be Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser,Ile, Gly, Val, Gln or Thr; Xaa18 can be His, Leu, Gln or Ala; Xaa19 canbe Pro, Gln, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile;Xaa31 can be Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32can be Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 canbe Ile, Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp orPhe; Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54can be any amino acid.

Additionally, each of the first four and at last three amino acids ofSEQ ID NO:1 can optionally be present or absent and can be any aminoacid, if present, e.g., any non-cysteine amino acid.

In one embodiment, the polypeptide has a sequence with one or more ofthe following properties: Xaa11 can be Asp, Gly, Ser or Val; Xaa13 canbe Pro, Arg, His or Asn; Xaa15 can be Arg or Lys; Xaa16 can be Ala orGly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18 can be His, Leu or Gln;Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trp or Phe; Xaa31 is Glu;Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr or Ser; Xaa35 is Tyr; andXaa39 can be Glu, Gly or Ala.

An exemplary polypeptide can include the following amino acids: Xaa10 isAsp; Xaa11 is Asp; Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can beAla or Gly; Xaa17 is Ala; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp;Xaa31 is Glu; Xaa32 is Glu; Xaa34 can be Ile or Ser; Xaa35 is Tyr; andXaa39 is Gly.

It is also possible to use portions of the polypeptides describedherein. For example, polypeptides could include binding domains forspecific kallikrein epitopes. For example, the binding loops of Kunitzdomains can by cyclized and used in isolation or can be grafted ontoanother domain, e.g., a framework of another Kunitz domain. It is alsopossible to remove one, two, three, or four amino acids from theN-terminus of an amino acid sequence described herein, and/or one, two,three, four, or five amino acids from the C-terminus of an amino acidsequence described herein.

Examples of sequences encompassed by SEQ ID NO:1 are described by thefollowing (where not indicated, “Xaa” refers to any amino acid, anynon-cysteine amino acid or any amino acid from the same set of aminoacids that are allowed for SEQ ID NO:1):

(SEQ ID NO:56) Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11 Gly Xaa13Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn Ile Phe Thr ArgGln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Gly Asn Gln AsnArg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp. Met His SerPhe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg TrpPhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys GluGly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr ArgAsp (amino acids 3-60 of SEQ ID NO:2), (SEQ ID NO:4) Met His Ser Phe CysAla Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Asn His Leu Arg Phe Phe PheAsn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly AsnGln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:5) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:6) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr Gly Gly Cys Ala Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:7) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys AlaSer Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe IleTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:8) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:9)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Gly Ala HisLeu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr GlyGly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:10) Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly Arg Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:11) Met HisSer Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Gly Ala His Pro ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:12) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:13) Met His Ser PheCys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala His Pro Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:14) Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg CysArg Gly Ala Gln Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:15) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Ser Cys Arg Ala Ala His Leu Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:16) Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Ser Cys Arg AlaAla His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:17) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Arg Gly Ala His Leu Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:18)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Arg Gly Ala LeuPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:19) Met His Ser Phe Cys Ala Phe Lys Ala AspSer Gly Asn Cys Arg Gly Asn Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:20) Met HisSer Phe Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg Gly Asn His Gln ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:21) Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly GlyArg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:22) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Arg Cys Arg Gly Ala His Pro Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.

Additional examples of sequence include those that differ by at leastone amino acid, but fewer than seven, six, five, four, three, or twoamino acids differences relative to an amino acid sequence describedherein, e.g., an amino acid sequence provided above. In one embodiment,fewer than three, two, or one differences are in one of the bindingloops. For example, the first binding loop may have no differencesrelative to an amino acid sequence described herein, e.g., an amino acidsequence provided above. In another example, neither the first nor thesecond binding loop differs from an amino acid sequence describedherein, e.g., an amino acid sequence provided above.

FIGS. 3A and 3B provides an amino acid sequence alignment of thesesequences, the native LACI sequence from which these variants werederived (SEQ ID NO:32), and other known Kunitz domains (SEQ ID NOS:29-31 and 33-53). Still others polypeptides that inhibit kallikreininclude an about 58-amino acid sequence of amino acids 3-60 of SEQ IDNO:2 or the PEP-1 polypeptide having the 60-amino acid sequence of SEQID NO:2. The term “PEP-1” and “DX-88” as used herein refer to the60-amino acid sequence of SEQ ID NO:2. A nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:2 is provided in SEQ ID NO:3 (see,e.g., nucleotides 309-488 in FIG. 2). It is understood that based on theknown genetic code, degenerate forms of the nucleotide sequence of SEQID NO:3 can be obtained by simply substituting one or more of the knowndegenerate codons for each amino acid encoded by the nucleotidesequence. Nucleotides 7-180 of SEQ ID NO:3, and degenerate formsthereof, encode the non-naturally occurring Kunitz domain polypeptidethat includes the 58-amino acid sequence of amino acids 3-60 of SEQ IDNO:2, a related sequence, or a functional fragment thereof.

In one embodiment, the polypeptide that inhibits kallikrein isaprotinin. In another embodiment, the polypeptide is other thanaprotinin, e.g., differs from aprotinin, by at least one, two, three,five, ten, or fifteen amino acids.

Polypeptides described herein can be made synthetically using anystandard polypeptide synthesis protocol and equipment. For example, thestepwise synthesis of a polypeptide can be carried out by the removal ofan amino (N) terminal-protecting group from an initial (i.e.,carboxy-terminal) amino acid, and coupling thereto of the carboxyl endof the next amino acid in the sequence of the polypeptide. This aminoacid is also suitably protected. The carboxyl group of the incomingamino acid can be activated to react with the N-terminus of the boundamino acid by formation into a reactive group such as formation into acarbodiimide, a symmetric acid anhydride, or an “active ester” groupsuch as hydroxybenzotriazole or pentafluorophenyl esters. Preferredsolid-phase peptide synthesis methods include the BOC method, whichutilizes tert-butyloxycarbonyl as the I-amino protecting group, and theFMOC method, which utilizes 9-fluorenylmethloxycarbonyl to protect theI-amino of the amino acid residues. Both methods are well known to thoseof skill in the art (Stewart, J. and Young, J., Solid-Phase PeptideSynthesis (W. H. Freeman Co., San Francisco 1989); Merrifield, J., 1963.Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky, A., ThePractice of Peptide Synthesis (Springer-Verlag, New York 1984)). Ifdesired, additional amino- and/or carboxy-terminal amino acids can bedesigned into the amino acid sequence and added during polypeptidesynthesis.

Polypeptides can also be produced using recombinant technology.Recombinant methods can employ any of a number of cells andcorresponding expression vectors, including but not limited to bacterialexpression vectors, yeast expression vectors, baculovirus expressionvectors, mammalian viral expression vectors, and the like. A polypeptidedescribed herein can be produced by a transgenic animal, e.g., in themammary gland of a transgenic animal. In some cases, it could benecessary or advantageous to fuse the coding sequence for a polypeptidethat inhibits kallikrein (e.g., a polypeptide that includes a Kunitzdomain) to another coding sequence in an expression vector to form afusion polypeptide that is readily expressed in a host cell. Part or allof the additional sequence can be removed, e.g., by protease digestion.

An exemplary recombinant expression system for producing a polypeptidethat inhibits kallikrein (e.g., a polypeptide that includes a Kunitzdomain) is a yeast expression vector, which permits a nucleic acidsequence encoding the amino acid sequence for the inhibitor polypeptideto be linked in the same reading frame with a nucleotide sequenceencoding the MAT® prepro leader peptide sequence of Saccharomycescerevisiae, which in turn is under the control of an operable yeastpromoter. The resulting recombinant yeast expression plasmid can betransformed by standard methods into the cells of an appropriate,compatible yeast host, which cells are able to express the recombinantprotein from the recombinant yeast expression vector. Preferably, a hostyeast cell transformed with such a recombinant expression vector is alsoable to process the fusion protein to provide an active inhibitorpolypeptide. An other exemplary yeast host for producing recombinantpolypeptides is Pichia pastoris.

As noted above, polypeptides that inhibit kallikrein can include aKunitz domain polypeptide described herein. Some polypeptides caninclude an additional flanking sequence, preferably of one to six aminoacids in length, at the amino and/or carboxy-terminal end, provided suchadditional amino acids do not significantly diminish kallikrein bindingaffinity or kallikrein inhibition activity so as to preclude use in themethods and compositions described herein. Such additional amino acidscan be deliberately added to express a polypeptide in a particularrecombinant host cell or can be added to provide an additional function,e.g., to provide a linker to another molecule or to provide an affinitymoiety that facilitates purification of the polypeptide. Preferably, theadditional amino acid(s) do not include cysteine, which could interferewith the disulfide bonds of the Kunitz domain.

An exemplary Kunitz domain polypeptide includes the amino acid sequenceof residues 3-60 of SEQ ID NO:2. When expressed and processed in a yeastfusion protein expression system (e.g., based on the integratingexpression plasmid pHIL-D2), such a Kunitz domain polypeptide retains anadditional amino terminal Glu-Ala dipeptide from the fusion with theMATalpha-prepro leader peptide sequence of S. cerevisiae. When secretedfrom the yeast host cell, most of the leader peptide is processed fromthe fusion protein to yield a functional polypeptide (referred to hereinas “PEP-1”) having the amino acid sequence of SEQ ID NO:2 (see boxedregion in FIG. 2).

In one embodiment, an inhibitor of kallikrein, e.g., a polypeptideinhibitor, has a binding affinity for kallikrein that is on the order of1000 times higher than that of aprotinin, which is currently approvedfor use in CABG procedures to reduce blood loss. The surprisingly highbinding affinities of such kallikrein inhibitors combined with theirhigh degree of specificity for kallikrein to the exclusion of othermolecular targets (see Table 1, below) provide for particularly usefulinhibitors. However, inhibitors with lesser affinity or specificity alsohave their applications.

A typical Kunitz domain, e.g., that includes, SEQ ID NO:1, contains anumber of invariant positions, e.g., positions corresponding to position5, 14, 30, 51 and 55 in the BPTI numbering scheme are cysteine. Thespacing between these positions may vary to the extent allowable withinthe Kunitz domain fold, e.g., such that three disulfide bonds areformed. Other positions such as, for example, positions 6, 7, 8, 9, 20,24, 25, 26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53 and 54,or positions corresponding to those positions, can be any amino acid(including non-naturally occurring amino acids). In a particularlypreferred embodiment, one or more amino acids correspond to that of anative sequence (e.g., SEQ ID NO:32, see FIG. 3). In another embodiment,at least one variable position is different from that of the nativesequence. In yet another preferred embodiment, the amino acids can eachbe individually or collectively substituted by a conservative ornon-conservative amino acid substitution.

Conservative amino acid substitutions replace an amino acid with anotheramino acid of similar chemical structure and may have no affect onprotein function. Non-conservative amino acid substitutions replace anamino acid with another amino acid of dissimilar chemical structure.Examples of conserved amino acid substitutions include, for example,Asn->Asp, Arg->Lys and Ser->Thr. In a preferred embodiment, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 ofthese amino acids can be independently or collectively, in anycombination, selected to correspond to the corresponding position of SEQID NO:2.

Other positions, for example, positions 10, 11, 13, 15, 16, 17, 18, 19,21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, or positionscorresponding to those positions can be any of a selected set of aminoacids. For example, SEQ ID NO: 1 defines a set of possible sequences.Each member of this set contains, for example, a cysteine at positions5, 14, 30, 51 and 55, and any one of a specific set of amino acids atpositions 10, 11, 13, 15, 16, 17, 18, 19, 221, 22, 23, 31, 32, 34, 35,39, 40, 43 and 45, or positions corresponding to those positions. In apreferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18 and/or 19 of these amino acids can be independently orcollectively, in any combination, selected to correspond to thecorresponding position of SEQ ID NO:2. The polypeptide preferably has atleast 80%, 85%, 90%, 95, 97, 98, or 99% identity to SEQ ID NO:2.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities. In the case of antibodies,the second antibody has the same specificity and has at least 50% of theaffinity of the same.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444-453, algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about what parameters should be applied todetermine if a molecule is within a homology limitation) are a Blossum62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4,and a frameshift gap penalty of 5.

Useful polypeptides can also be encoded by a nucleic acid thathybridizes to a nucleic acid that encodes a polypeptide describedherein. The nucleic acids can hybridize under medium, high, or very highstringency conditions. As used herein, the term “hybridizes under lowstringency, medium stringency, high stringency, or very high stringencyconditions” describes conditions for hybridization and washing. Guidancefor performing hybridization reactions can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, whichis incorporated by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridizationconditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C.

Kallikrein Inhibitors—Antibodies

One class of kallikrein inhibitors includes antibodies. Exemplaryantibodies bind, e.g., specifically to kallikrein, e.g., plasmakallikrein. An antibody can inhibit kallikrein in a number of ways. Forexample, it can contact one or more residues of the active site,sterically hinder or obstruct access to the active site, preventmaturation of kallikrein, or destabilize a conformation required forcatalytic activity.

As used herein, the term “antibody” refers to a protein that includes atleast one immunoglobulin variable domain or immunoglobulin variabledomain sequence. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab fragments,F(ab′)₂, a Fd fragment, a Fv fragments, and dAb fragments) as well ascomplete antibodies.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDR's has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917). Kabat definitions are usedherein. Each VH and VL is typically composed of three CDR's and fourFR's, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules. Immunoglobulin domainstypically contain two β-sheets formed of about seven β-strands, and aconserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay1988 Ann. Rev Immunol. 6:381-405).

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that includes immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that preferentially interacts with anactivated integrin structure or a mimic of an activated integrinstructure, e.g., relative to an non-activated structure.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. The heavy chain constantregion includes three domains, CH1, CH2 and CH3. The light chainconstant region includes a CL domain. The variable region of the heavyand light chains contains a binding domain that interacts with anantigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term “antibody”includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (aswell as subtypes thereof). The light chains of the immunoglobulin may beof types kappa or lambda. In one embodiment, the antibody isglycosylated. An antibody can be functional or non-functional forantibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human.For example, one or more of the variable regions can be human oreffectively human. For example, one or more of the CDRs can be human,e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each ofthe light chain CDRs can be human. HC CDR3 can be human. One or more ofthe framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of theHC or LC. In one embodiment, all the framework regions are human, e.g.,derived from a human somatic cell, e.g., a hematopoietic cell thatproduces immunoglobulins or a non-hematopoietic cell. In one embodiment,the human sequences are germline sequences, e.g., encoded by a germlinenucleic acid. One or more of the constant regions can be human oreffectively human. In another embodiment, at least 70, 75, 80, 85, 90,92, 95, or 98% of, or the entire antibody can be human or effectivelyhuman. An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 Kd or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH—terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

One exemplary method for identifying antibodies that bind to and inhibitkallikrein includes immunizing a non-human animal with kallikrein or afragment thereof. Even small peptides can be used as immunogens. In oneembodiment, a mutated kallikrein which has reduced or no catalyticactivity is used as immunogen. Spleen cells can be isolated from theimmunized animal and used to produce hybridoma cells using standardmethods. In one embodiment, the non-human animal includes one or morehuman immunoglobulin genes.

Another exemplary method for identifying proteins that bind to andinhibit kallikrein includes: providing a library of proteins andselecting from the library one or more proteins that bind to akallikrein or a fragment thereof. The selection can be performed in anumber of ways. For example, the library can be provided in the formatof a display library or a protein array. Prior to selecting, the librarycan be pre-screened (e.g., depleted) to remove members that interactwith a non-target molecule, e.g., protease other than a kallikrein or akallikrein in which the active site is inaccessible, e.g., bound by aninhibitor, e.g., aprotinin.

Antibody libraries, e.g., antibody display libraries, can be constructedby a number of processes (see, e.g., de Haard et al. (1999) J. Biol.Chem. 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20.and Hoogenboom et al. (2000) Immunol Today 21:371-8). Further, elementsof each process can be combined with those of other processes. Theprocesses can be used such that variation is introduced into a singleimmunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulindomains (e.g., VH and VL). The variation can be introduced into animmunoglobulin variable domain, e.g., in the region of one or more ofCDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4, referring to such regions ofeither and both of heavy and light chain variable domains. In oneembodiment, variation is introduced into all three CDRs of a givenvariable domain. In another preferred embodiment, the variation isintroduced into CDR1 and CDR2, e.g., of a heavy chain variable domain.Any combination is feasible.

In an exemplary system for recombinant expression of an antibody (e.g.,a full length antibody or an antigen-binding portion thereof), arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells, and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G. Antibodies can also beproduced by a transgenic animal.

Kallikrein Inhibitors—Peptides

The binding ligand can include a peptide of 32 amino acids or less thatindependently binds to a target molecule. Some such peptides can includeone or more disulfide bonds. Other peptides, so-called “linearpeptides,” are devoid of cysteines. In one embodiment, the peptides areartificial, i.e., not present in a Nature or not present in a proteinencoded by one or more genomes of interest, e.g., the human genome.Synthetic peptides may have little or no structure in solution (e.g.,unstructured), heterogeneous structures (e.g., alternative conformationsor “loosely structured), or a singular native structure (e.g.,cooperatively folded). Some synthetic peptides adopt a particularstructure when bound to a target molecule. Some exemplary syntheticpeptides are so-called “cyclic peptides” that have at least a disulfidebond and, for example, a loop of about 4 to 12 non-cysteine residues.Exemplary peptides are less than 28, 24, 20, or 18 amino acids inlength.

Peptide sequences that independently bind kallkrein can be identified byany of a variety of methods. For example, they can be selected from adisplay library or an array of peptides. After identification, suchpeptides can be produced synthetically or by recombinant means. Thesequences can be incorporated (e.g., inserted, appended, or attached)into longer sequences.

The following are some exemplary phage libraries that can be screened tofind at least some of the peptide ligands described herein. Each librarydisplays a short, variegated exogenous peptide on the surface of M13phage. The peptide display of five of the libraries was based on aparental domain having a segment of 4, 5, 6, 7, 8, 10, 11, or 12 aminoacids, respectively, flanked by cysteine residues. The pairs ofcysteines are believed to form stable disulfide bonds, yielding a cyclicdisplay peptide. The cyclic peptides are displayed at the amino terminusof protein III on the surface of the phage. The libraries weredesignated TN6/7, TN7/4, TN8/9, TN9/4, TN10/10. TN11/1, and TN12/1. Aphage library with a 20-amino acid linear display was also screened;this library was designated Lin20.

The TN6/7 library was constructed to display a single cyclic peptidecontained in a 12-amino acid variegated template. The TN6/6 libraryutilized a template sequence ofXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Cys₉-Xaa₁₀-Xaa₁₁-Xaa₁₂, whereeach variable amino acid position in the amino acid sequence of thetemplate is indicated by a subscript integer. Each variable amino acidposition (Xaa) in the template was varied to contain any of the commonα-amino acids, except cysteine (Cys).

The TN7/4 library was constructed to display a single cyclic peptidecontained in a 12-amino acid variegated template. The TN7/4 libraryutilized a template sequence ofXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃,where each variable amino acid position in the amino acid sequence ofthe template is indicated by a subscript integer. Each variable aminoacid position (Xaa) in the template was varied to contain any of thecommon α-amino acids, except cysteine (Cys).

The TN8/9 library was constructed to display a single binding loopcontained in a 14-amino acid template. The TN8/9 library utilized atemplate sequence ofXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Cys-Xaa₁₂-Xaa₁₃-Xaa₁₄.Each variable amino acid position (Xaa) in the template were varied topermit any amino acid except cysteine (Cys).

The TN9/4 library was constructed to display a single binding loopcontained in a 15-amino acid template. The TN9/4 library utilized atemplate sequenceXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa11-Cys₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅.Each variable amino acid position (Xaa) in the template were varied topermit any amino acid except cysteine (Cys).

The TN10/10 library was constructed to display a single cyclic peptidecontained in a 16-amino acid variegated template. The TN10/9 libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Cys₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆,where each variable amino acid position in the amino acid sequence ofthe template is indicated by a subscript integer. Each variable aminoacid position (Xaa) was to permit any amino acid except cysteine (Cys).

The TN11/1 library was constructed to display a single cyclic peptidecontained in a 17-amino acid variegated template. The TN11/1 libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa11-Xaa₁₂-Xaa₁₃-Cys14-Xaa₁₅-Xaa₁₆-Xaa₁₇, where each variable amino acid position in theamino acid sequence of the template is indicated by a subscript integer.Each variable amino acid position (Xaa) was to permit any amino acidexcept cysteine (Cys).

The TN12/1 library was constructed to display a single cyclic peptidecontained in an 18-amino acid template. The TN12/1 library utilized atemplate sequenceXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa8-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Cys15-Xaa₁₆-Xaa₁₇-Xaa₁₈, where each variable amino acid position in theamino acid sequence of the template is indicated by a subscript integer.The amino acid positions Xaa₁, Xaa₂, Xaa₁₇ and Xaa₁₈ of the templatewere varied, independently, to permit each amino acid selected from thegroup of 12 amino acids consisting of Ala, Asp, Phe, Gly, His, Leu, Asn,Pro, Arg, Ser, Trp, and Tyr. The amino acid positions Xaa₃, Xaa₅, Xaa₆,Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa11, Xaa12, Xaa13, Xaa14, Xaa16, of thetemplate were varied, independently, to permit any amino acid exceptcysteine (Cys).

The Lin20 library was constructed to display a single linear peptide ina 20-amino acid template. The amino acids at each position in thetemplate were varied to permit any amino acid except cysteine (Cys).

The techniques discussed in Kay et al., Phage Display of Peptides andProteins: A Laboratory Manual (Academic Press, Inc., San Diego 1996) andU.S. Pat. No. 5,223,409 are useful for preparing a library of potentialbinders corresponding to the selected parental template. The librariesdescribed above can be prepared according to such techniques, andscreened, e.g., as described above, for peptides that bind to andinhibit kallikrein.

In addition phage libraries or selected populations from phage librariescan be counter-selected, e.g., using kallikrein that is inactivated,e.g., by binding of aprotinin or another kallikrein inhibitor. Suchprocedures can be used to discard peptides that do not contact theactive site.

Peptides can also be synthesized using alternative backbones, e.g., apeptoid backbone, e.g., to produce a compound which has increasedprotease resistance. In particular this method can be used to make acompound that binds to and inhibits kallikrein and which is not itselfeffectively cleaved by kallikrein.

Modifications

It is possible to modify polypeptides that inhibit a Kunitz domain in avariety of ways. For example, the polypeptides can be attached to one ormore polyethylene glycol moieties to stabilize the compound or prolongretention times, e.g., by at least two, four, five, or eight fold.

A polypeptide that inhibits kallikrein can be associated with (e.g.,conjugated to) a polymer, e.g., a substantially non-antigenic polymers,such as polyalkylene oxides or polyethylene oxides. Suitable polymerswill vary substantially by weight. Polymers having molecular numberaverage weights ranging from about 200 to about 35,000 (or about 1,000to about 15,000, and 2,000 to about 12,500) can be used. A plurality ofpolymer moieties can be attached to one polypeptide, e.g., at least two,three, or four such moieties, e.g., having an average molecular weightof about 2,000 to 7,000 Daltons.

For example, the polypeptide can be conjugated to a water solublepolymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol andpolyvinylpyrrolidone. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained. Additional useful polymers includepolyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and blockcopolymers of polyoxyethylene and polyoxypropylene (Pluronics);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextranesulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; heparin or heparon.

Other compounds can also be attached to the same polymer, e.g., acytotoxin, a label, or another targeting agent or an unrelated agent.Mono-activated, alkoxy-terminated polyalkylene oxides (PAO's), e.g.,monomethoxy-terminated polyethylene glycols (mPEG's); C₁₋₄alkyl-terminated polymers; and bis-activated polyethylene oxides(glycols) can be used for crosslinking. See, e.g., U.S. Pat. No.5,951,974

Kallikrein Inhibitors—Small Molecules

An inhibitor of a kallikrein, e.g., plasma kallikrein, can also be acompound that is smaller than 3000, 2000 or 1000 Daltons. For example,the compound is a non-proteinaceous compound or a compound that includesfewer than five peptide bonds.

For example, the inhibitor can be a compound described in WO 04/062657,e.g., an acylated 4-amidino- or 4-guanidinobenzylamine. The compound canhave the general formula (I) P4-P3-P2-P1 (I), where P4 is a mono- orpoly-substituted or unsubstituted benzylsulphonyl group, P3 is a mono-or poly-substituted or unsubstituted, natural or unnatural alpha-aminoor alpha-imino acid with the D-configuration, P2 is a mono- orpoly-substituted or unsubstituted natural or unnatural alpha-amino oralpha-imino acid with the L-configuration and P1 is a mono- orpoly-substituted or unsubstituted 4-amidino- or 4-guanidinobenzylaminegroup.

Another example is a compound represented by Structural Formula (I):

R1 is a substituted or unsubstituted aryl group or alkyl group; R2 is asubstituted or unsubstituted aryl group or cycloalkyl group; Ar is asubstituted or unsubstituted aryl group; X is a —CH2-, —O—, —S— or —CO—;m is an integer from zero to two; n is an integer from 0-2 when X is—O—, —S— and 1-2 when X is —CH2- or —CO—. Exemplary compounds aredescribed in US 2004-044075.

Methods and Compositions

The inhibitors described herein can be used in methods for preventing orreducing ischemia and/or reperfusion injury associated with ischemia;methods for preventing or reducing perioperative blood loss and/or asystemic inflammatory response (SIR) in a patient, especially associatedwith cardiothoracic surgery. The method includes administering ainhibitor of kallikrein, e.g., plasma kallikrein.

In one embodiment, a method for treatment involves the administration ofa polypeptide comprising a Kunitz domain. One embodiment of the methodinvolves using a polypeptide containing an amino acid sequence of SEQ IDNO:1 that has an affinity for kallikrein that is approximately 1000-foldor more higher than that of a broad range serine protease, e.g.,aprotinin, which is isolated from bovine lung and currently approved foruse in CABG procedures (TRASYLOL™, Bayer Corporation PharmaceuticalDivision, West Haven, Conn.).

Patients subjected to any of a number of surgical procedures, especiallythose involving extra-corporeal circulation, e.g., cardiothoracicsurgery, such as, for example, CPB, and/or bone trauma, such as sternalsplit or hip replacement, are at risk for perioperative blood loss andinflammation. Contact of a patient's blood with the cut surfaces of boneor of CPB equipment is sufficient to activate one or several undesirablecascade responses, including a contact activation system (CAS), whichcan lead to extensive perioperative blood loss requiring immediate bloodtransfusion, as well as a systemic inflammatory response (SIR), which,in turn, can result in permanent damage to tissues and organs. While notdesiring to be limited to any particular mechanism or theory, it appearsthat the blood loss that occurs associated with cardiothoracic surgery,e.g., CPB, as in a CABG procedure, probably results from extensivecapillary leakage, which can result in significant loss of blood thatmust be replaced by immediate blood transfusion.

The inhibitors described herein can be used to prevent or reduce variousischemias including, for example, perioperative blood loss and SIR in apatient subjected to a surgical procedure, and especially wherein thesurgical procedure requires extra-corporeal circulation, e.g.,cardiothoracic surgery, such as, for example, CPB. The inhibitors can beparticularly useful for preventing or reducing perioperative blood lossand/or SIR in a patient subjected to a CABG procedure requiring CPB orother cardiac surgery. Further, the inhibitors described herein can beused to prevent or reduce cerebral ischemia (such as stroke) and/orreperfusion injury associated with cerebral ischemia (e.g., stroke).

Exemplary compositions for medical use comprise a kallikrein inhibitordescribed herein. Such compositions can further include one or morepharmaceutically acceptable buffers, carriers, and excipients, which canprovide a desirable feature to the composition including, but notlimited to, enhanced administration of the composition to a patient,enhanced circulating half-life of the inhibitor, enhanced compatibilityof the inhibitor with patient blood chemistry, enhanced storage of thecomposition, and/or enhanced delivery and/or efficacy of the inhibitorupon administration to a patient. In addition to an inhibitor describedherein, compositions can further include one or more otherpharmaceutically active compounds that provide an additionalprophylactic or therapeutic benefit to a patient, e.g., a patient of aninvasive surgical procedure or a patent otherwise at risk for, having orpreviously had cerebral ischemia and/or reperfusion injury associatedwith cerebral ischemia. For example, the compositions can includeanother compound described herein.

Perioperative Blood Loss and Reduced Heart Bloodflow

Due to the many advances in medicine, a number of highly invasivesurgical procedures are carried out each day that result in blood loss,or place patients at a high risk for blood loss. Such patients aregenerally carefully monitored to restore and maintain normal bloodsupply and hemostasis, and they may need blood transfusions. Surgicalprocedures that involve blood loss include those involvingextra-corporeal circulation methods such as cardiothoracic surgery,e.g., CPB. In such methods, a patient's heart is stopped and thecirculation, oxygenation, and maintenance of blood volume are carriedout artificially using an extra-corporeal circuit and a syntheticmembrane oxygenator. These techniques are commonly used during cardiacsurgery. Additionally, it is apparent that surgery involving extensivetrauma to bone, such as the sternal split necessary in CABG or hipreplacement procedures, is also associated with activation of the CAS,which can result in a variety of disruptions in the blood andvasculature.

Atherosclerotic coronary artery disease (CAD) causes a narrowing of thelumen of one or several of the coronary arteries; this limits the flowof blood to the myocardium (i.e., the heart muscle) and can causeangina, heart failure, and myocardial infarcts. In the end stage ofcoronary artery atherosclerosis, the coronary circulation can be almostcompletely occluded, causing life threatening angina or heart failure,with a very high mortality. CABG procedures may be required to bridgethe occluded blood vessel and restore blood to the heart; these arepotentially life saving. CABG procedures are among the most invasive ofsurgeries in which one or more healthy veins or arteries are implantedto provide a “bypass” around the occluded area of the diseased vessel.CABG procedures carry with them a small but important perioperativerisk, but they are very successful in providing patients with immediaterelief from the mortality and morbidity of atheroscleroticcardiovascular disease. Despite these very encouraging results, repeatCABG procedures are frequently necessary, as indicated by an increase inthe number of patients who eventually undergo second and even thirdprocedures; the perioperative mortality and morbidity seen in primaryCABG procedures is increased in these re-do procedures.

There have been improvements in minimally invasive surgical techniquesfor uncomplicated CAD. However, nearly all CABG procedures performed forvalvular and/or congenital heart disease, heart transplantation, andmajor aortic procedures, are still carried out on patients supported byCPB. In CPB, large cannulae are inserted into the great vessels of apatient to permit mechanical pumping and oxygenation of the blood usinga membrane oxygenator. The blood is returned to the patient withoutflowing through the lungs, which are hypoperfused during this procedure.The heart is stopped using a cardioplegic solution, the patient cooledto help prevent brain damage, and the peripheral circulating volumeincreased by an extracorporeal circuit, i.e., the CPB circuit, whichrequires “priming” with donor blood and saline mixtures are used to fillthe extracorporeal circuit. CPB has been extensively used in a varietyof procedures performed for nearly half a century with successfuloutcomes. The interaction between artificial surfaces, blood cells,blood proteins, damaged vascular endothelium, and extravascular tissues,such as bone, disturbs hemostasis and frequently activates the CAS,which, as noted above, can result in a variety of disruptions in theblood and vasculature. Such disruption leads to excess perioperativebleeding, which then requires immediate blood transfusion. A consequenceof circulating whole blood through an extracorporeal circuit in CPB canalso include the systemic inflammatory response (SIR), which isinitiated by contact activation of the coagulation and complementsystems. Indeed, much of the morbidity and mortality associated withseemingly mechanically successful CPB surgical procedures is the resultof the effects of activating coagulation, fibrinolysis, or complementsystems. Such activation can damage the pulmonary system, leading toadult respiratory distress syndrome (ARDS), impairment of kidney andsplanchnic circulation, and induction of a general coagulopathy leadingto blood loss and the need for transfusions. In addition to the dangersof perioperative blood loss, additional pathologies associated with SIRinclude neurocognitive deficits, stroke, renal failure, acute myocardialinfarct, and cardiac tissue damage.

Blood transfusions also present a significant risk of infection andelevate the cost of CABG or other similar procedures that require CPB.In the absence of any pharmacological intervention, three to seven unitsof blood must typically be expended on a patient, even with excellentsurgical techniques. Accordingly, there is considerable incentive forthe development of new and improved pharmacologically effectivecompounds to reduce or prevent perioperative bleeding and SIR inpatients subjected to CPB and CABG procedures. Use of the inhibitorsdescribed herein can improve these various treatments and lead toamelioration of the undesirable symptoms that can occur.

Cerebral Ischemia and Reperfusion Injury

The methods described herein are useful for reducing or preventingcerebral ischemia as well as reperfusion injury associated with cerebralischemia. A “cerebral ischemic attack” or “cerebral ischemia” is anischemic condition in which blood supply to the brain is blocked. Thisinterruption in the blood supply to the brain may result from a varietyof causes including, but not limited to, an intrinsic blockage orocclusion of the blood vessel itself, a remotely originated source ofocclusion, decreased perfusion pressure or increased blood viscosityresulting in decreased cerebral blood flow, or ruptured or leaky bloodvessels in the subarachnoid space or intracerebral tissue. Cerebralischemia may result in either transient or permanent deficits and theseriousness of the neurological damage in a patient who has experiencedcerebral ischemia depends on the intensity and duration of the ischemiaevent. A transient ischemia attack (TIA) is one in which the blood flowto the brain is briefly interrupted and causes temporary neurologicaldeficits. Symptoms of TIA include numbness of weakness of face or limbs,loss of ability to speak clearly and/or understand the speech of others,a loss of vision or dimness of vision and dizziness. Permanent cerebralischemia attacks, also called strokes, are caused by a longerinterruption in blood flow to the brain resulting from an embolism, athrombus or bleeding in the brain (e.g., a hemorrhage). The term“thromboembolic stroke” or “thromboembolism” is used herein to refer toa stroke caused by either a thrombosis or an embolism. A stroke causes aloss of neurons typically resulting in a neurological deficit that mayimprove but does not entirely resolve. The inhibitors described hereinare useful in preventing or reducing stroke including embolic-,thrombolic-, thromboembolic- and hemorrhage-associated strokes. Strokescan be caused by a variety of causes. One category includesperioperative strokes that can be associated with thrombus or embolismformation.

In stroke patients, there is a core of the neurological deficit markedby total ischemia and/or tissue necrosis. This area is normallysurrounded by ischemic tissue, referred to as the ischemic penumbra,that receives collateral circulation. Ischemia in the penumbra does notalways result in irreversible damage. In some cases, restoration ofblood flow (reperfusion) into the penumbra may prevent total ischemiaand necrosis in this area. However, reperfusion has also been associatedwith injury to the tissue surrounding the core. Once blood flow isreturned, blood cells such as neutrophils, attack the damaged tissuewhich can cause additional inflammation and/or damage. Reperfusioninjury is associated with an influx of neutrophils into the affectedtissue and subsequent activation of the neutrophils. Neutrophils canrelease lytic enzymes that directly induce tissue damage andproinflammatory mediators such as cytokines that amplify localinflammatory reaction. The influx of neutrophils to a site of ischemicdamage can also plug capillaries and cause vasoconstriction. It has beenfound that kallikrein plays a role in neutrophil chemotaxis, neutrophilactivation and reperfusion injury. Thus, the kallikrein inhibitorsdescribed herein can be used to prevent or reduce reperfusion injury,e.g., by reducing or preventing one or more of: 1) neutrophilinfiltration, 2) neutrophil activation; 3) cytokine release; 4) elastaserelease; and 5) vasodilation. For example, a kallikrein inhibitor can beused to inhibit bradykinin and Factor XII.

Administration

A kallikrein inhibitor can be administered to a patient before, during,and/or after an ischemia event, e.g., a surgical procedure or cerebralischemic attack, in a pharmaceutically acceptable composition or inconnection with another disorder or event described herein. The patientis generally a human, but may also be a non-human mammal. Human patientsinclude adults, e.g., patients between ages 19-25, 26-40, 41-55, 56-75,and 76 and older, and pediatric patients, e.g., patients between ages0-2, 3-6, 7-12, and 13-18.

The term “pharmaceutically acceptable” composition refers to a non-toxiccarrier or excipient that may be administered to a patient, togetherwith a kallikrein inhibitor described herein. The carrier or excipientis chosen to be compatible with the biological or pharmacologicalactivity of the composition. The inhibitors described herein can beadministered locally or systemically by any suitable means for deliveryof a kallikrein inhibitory amount of the inhibitor to a patientincluding but not limited to systemic administrations such as, forexample, intravenous and inhalation. Parenteral administration isparticularly preferred.

For parenteral administration, the polypeptides can be injectedintravenously, intramuscularly, intraperitoneally, or subcutaneously.Intravenous administration is preferred. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Other pharmaceutically acceptable carriers include, but are notlimited to, sterile water, saline solution, and buffered saline(including buffers like phosphate or acetate), alcohol, vegetable oils,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, paraffin, etc. Where necessary, the composition canalso include a solubilizing agent and a local anaesthetic such aslidocaine to ease pain at the site of the injection, preservatives,stabilizers, wetting agents, emulsifiers, salts, lubricants, etc. aslong as they do not react deleteriously with the active compounds.Similarly, the composition can comprise conventional excipients, e.g.,pharmaceutically acceptable organic or inorganic carrier substancessuitable for parenteral, enteral or intranasal application which do notdeleteriously react with the active compounds. Generally, theingredients will be supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent in activity units.Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade “water for injection” or saline. Where the composition is to beadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

In one embodiment, the inhibitor is administered to a patient as anintravenous infusion according to any approved procedure. For example,an inhibitor described herein can be administered to a patient subjectedto a CABG procedure at the times similar to those currently used inapproved protocols for administering aprotinin and in an amountnecessary to provide a patient with a required number or concentrationof kallikrein inhibitory units (KIU).

An inhibitor described herein can also be administered to a patient inthe immediate postoperative period, when bleeding abnormalities canoccur as a consequence of downstream effects of SIR. For example, in aprocedure involving CPB, an inhibitor described herein can beadministered to a patient as an initial loading dose, e.g., an effectiveamount over the course of a convenient time, such as 10 minutes, priorto induction of anesthesia. Then, at induction of anesthesia, a seconddose of the inhibitor can be injected into the CPB priming fluid (“pumpprime volume”). The patient can then be placed on a continuous andcontrolled intravenous infusion dose for the duration of the surgicalprocedure, and after the procedure if indicated.

In other embodiments, an inhibitor can be administered after an ischemicevent, e.g., after a stroke, e.g., 5, 10, 15, 30, 45 minutes, 1, 2, 3,5, 10, 15, 20 hours or more after a stroke. Preferably, the inhibitor isadministered within 12 to 60 hours, e.g., within 24 to 48 hours, after astroke. In some embodiments, an inhibitor is administered after anischemic event, e.g., after a stroke, but prior to reperfusion of thedamaged tissue. In other embodiments, an inhibitor is administeredduring reperfusion or after reperfusion has begun. In yet anotherembodiment, an inhibitor is administered after reperfusion has occurred.

An effective amount of an inhibitor (e.g., a Kunitz domain polypeptideor other compound described herein) can be administered alone or incombination with another therapeutic for the treatment of cerebralischemia and/or reperfusion injury associated with cerebral ischemia. Aneffective amount is an amount sufficient to reduce one or more symptomsassociated with cerebral ischemia and/or reperfusion injury associatedwith cerebral ischemia which otherwise would have occurred in a subjectexperiencing a cerebral ischemia and/or reperfusion injury associatedwith cerebral ischemia absent the treatment. Several physiologicalparameters may be used to assess stroke and reperfusion injuryassociated with stroke including infarct size, regional cerebral bloodflow, intracranial pressure, anterograde amnesia, retrograde amnesia,dementia, cognitive function and/or emotion, and cerebral edema, forexample, as compared to pretreatment patient parameters, untreatedstroke patients or stroke patients treated with the other therapeuticagent but not the combination with the inhibitor (e.g., the Kunitzdomain polypeptide or other compound described herein).

With respect to an implementation in which DX-88 or a DX-88-relatedinhibitor is used, the affinity constant (Ki) of DX-88 is at least about1000 times greater than aprotinin for kallikrein inhibition.Accordingly, the dose of DX-88 or an inhibitor of similar affinity couldbe at least about 10, 50, 100, 500, or 1000 times lower than aprotininon a mole per mole basis. The dose could also be modulated as a functionof the amount of kallikrein activated during an event (e.g., CPB), thespecificity of the DX-88-kallikrein interaction in vivo, theconcentration of kallikrein eliciting SIRS, and pharmacologicaldistribution.

The total amount of circulating prekallikrein in plasma is reported tobe approximately 50 g/mL or 500 nM. If all prekallikrein is activated,at least 500 nmoles/L of DX-88 can be used to inhibit kallikrein in astoichiometric manner. An individual having 5 L of plasma would requirea dose of 2500 nmoles DX-88, or approximately 18 mg based on themolecular weight of DX-88 of 7,054 Daltons. The dose can be reducedproportionally if not all of the prekallikrein is activated.

As the concentration of active kallikrein may have to rise above acertain level to contribute to increased fluid and blood losspost-operatively, in many cases, it is not necessary to inactivate allactive kallikrein. DX-88 would be expected to be effective at asignificantly lower dose compared to aprotinin on the basis of itshigher affinity for kallikrein. A plasma level of 10 nM DX-88 wouldinhibit 99.6% of the kallikrein present at a plasma concentration of 1nM (i.e., only 0.4 μM free kallikrein remaining), while only 24.5% ofthe activated kallikrein would be inhibited by 10 nM aprotinin. Thesevalues were calculated using the standard equilibrium equation thatrelates the Ki of the binding interaction to the relative concentrationof enzyme (plasma kallikrein) and inhibitor (DX-88). Plasma levels of atleast 3 μM aprotinin would be required to inhibit kallikrein to the sameextent as 10 nM DX-88. In other words, the high affinity of DX-88 forkallikrein can allow for a rapid therapeutic effect without maintaininghigh plasma inhibitor levels.

DX-88 also has greater specificity for kallikrein inhibition compared toaprotinin in vitro. Therefore, proteases other than kallikrein that areinhibited by aprotinin may lower the effective concentration of theinhibitor, thereby increasing the amount of aprotinin needed for atherapeutic effect.

An initial clinical dose of DX-88 for patients undergoing CPB has beenestimated from the recommended high dose regimen of aprotinin (2×10⁶KIU). Aprotinin is administered in three stages of 2×10⁶ KIU each,consisting of a loading dose, a pump priming dose and a continuousintravenous infusion during CPB. Aprotinin is reported to have aspecific inhibitory activity of 7,143 KIU/mgv, determined using a dogblood pressure assay. Therefore, 2×10⁶ KIU of aprotinin is equivalent to280 mg of the protein. In a patient having a plasma volume of 5 liters,280 mg corresponds to approximately 8.6 μM aprotinin. The specificactivity of aprotinin in the inhibitory assay used for DX-88 isapproximately 0.5 KIU/mg determined using an enzymatic assay. A dose of280 mg would correspond to a loading dose for aprotinin of approximately140 KIU. The specific activity of DX-88 using the same assay isapproximately 10 KIU/mg. A dose of only 14 mg of DX-88 would be requiredto provide a number of inhibitory units equivalent to 280 mg aprotinin.Thus, in certain embodiments, about 5-20, or 10-20 mg of DX-88 can beadministered to a subject.

Administered “in combination,” as used herein, means that two (or more)different treatments are delivered to the subject once the subject hasbeen determined to be at risk for the disorder or during the course ofthe subject's affliction with the disorder, e.g., the two or moretreatments are delivered after the subject has been diagnosed as at riskfor the disorder or the two or more agents are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated. In some embodiments, the delivery of onetreatment is still occurring when the delivery of the second begins, sothat there is overlap. This is sometimes referred to herein as“simultaneous” or “concurrent delivery.” In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered. The Kunitz domain polypeptide or other inhibitor can beadministered before, concurrently with, or after the administration ofanother therapeutic, e.g., an anticoagulant agent, an antiplatelet agentor a thrombolytic agent.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, hirudin, bivalarutin, and otherdirect thrombin inhibitors, and indandione derivatives.

Anti-platelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Anti-platelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetics.

Thrombolytic agents lyse clots that cause the thromboembolic stroke.Thrombolytic agents have been used in the treatment of acute venousthromboembolism and pulmonary emboli and are well known in the art (e.g.see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S(1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p. 10S-17S(1995)). Thrombolytic agents include, but are not limited to,plasminogen, a2-antiplasmin, streptokinase, antistreplase, TNK, tissueplasminogen activator (tPA), and urokinase. “tPA” as used hereinincludes native tPA and recombinant tPA, as well as modified forms oftPA that retain the enzymatic or fibrinolytic activities of native tPA.The enzymatic activity of tPA can be measured by assessing the abilityof the molecule to convert plasminogen to plasmin. The fibrinolyticactivity of tPA may be determined by any in vitro clot lysis activityknown in the art, such as the purified clot lysis assay described byCarlson, et al., Anal. Biochem. 168, 428-435 (1988) and its modifiedform described by Bennett, W. F. et al., 1991, supra.

Currently there are two regimens approved in the United States foradministering aprotinin to a patient undergoing a CABG procedure (see,product label and insert for TRASYLOL™, Bayer Corporation PharmaceuticalDivision, West Haven, Conn.). One such approved regimen uses a 2 millionKIU intravenous loading dose, 2 million KIU into the pump prime volume,and 500,000 KIU per hour of surgery. Another approved regimen uses 1million KIU intravenous loading dose, 1 million KIU into the pump primevolume, and 250,000 KIU per hour of surgery. As these regimens are basedon KIU, the regimens are readily adapted to other kallikrein inhibitorsdescribed herein once the specific activity and KIU of a particularinhibitor has been determined by standard assays. Owing to the enhancedbinding affinity and inhibitory activity in representative polypeptideinhibitors of kallikrein described herein relative to aprotinin, it isexpected that the compositions and methods described herein are likelyto require fewer milligrams (mg) per patient to provide a patient withthe required number or concentration of KIU.

Several considerations regarding dosing with a polypeptide inhibitor ofkallikrein can be illustrated by way of example with the representativePEP-1 KI polypeptide having the amino sequence of SEQ ID NO:2 (molecularweight of 7,054 Daltons).

Table 1, below, provides a comparison of the affinity (Ki,app) of thePEP-1 KI polypeptide for kallikrein and eleven other known plasmaproteases.

TABLE 1 PEP-1 K_(i), Aprotinin K_(i), Protease Substrate app (pM) app(pM) human plasma kallikrein 44  3.0 × 10⁴ human urine kallikrein  >1 ×10⁸  4.0 × 10³ porcine pancreatic  2.7 × 10⁷ 550 kallikrein human C1r,activated >2.0 × 10⁸ >1.0 × 10⁷ human C1s, activated >2.0 × 10⁷ >1.0 ×10⁸ human plasma factor XIa  1.0 × 10⁴ ND human plasma factor XIIa >2.0× 10⁷ >1.0 × 10⁸ human plasmin  1.4 × 10⁵ 894 human pancreatic trypsin >2 × 10⁷ ND human pancreatic >2.0 × 10⁷  7.3 × 10⁵ chymotrypsin humanneutrophil elastase >2.0 × 10⁷  1.7 × 10⁶ human plasma thrombin >2.0 ×10⁷ >1.0 × 10⁸ ND = not determined

Clearly, the PEP-1 KI polypeptide is highly specific for human plasmakallikrein. Furthermore, the affinity (K_(i),app) of PEP-1 forkallikrein is 1000 times higher than the affinity of aprotinin forkallikrein: the K_(i),app of PEP-1 for kallikrein is about 44 pM (Table1), whereas the K_(i),app of aprotinin for kallikrein is 30,000 pM.Thus, a dose of PEP-1 could be approximately 1000 times lower than thatused for aprotinin on a per mole basis. However, consideration ofseveral other factors may provide a more accurate estimation of the doseof PEP-1 required in practice. Such factors include the amount ofkallikrein activated during CPB in a particular patient, theconcentration of kallikrein required to elicit an SIR, and thebioavailability and pharmacological distribution of PEP-1 in a patient.Nevertheless, use of a polypeptide that includes a Kunitz domain thatinhibits kallikrein in doses currently approved for the use of aprotininis still expected to provide significant improvements over the currentuse of the less specific, lower affinity, bovine aprotinin. Accordingly,lower doses, e.g., at least half, or a tenth of the approved aprotinindose may be used for a kallikrein inhibitor which inhibits kallikrein atleast 2, 10, 50, or 100 fold better than aprotinin.

For example, the total amount of circulating prekallikrein in plasma isestimated at approximately 500 nM (Silverberg, M. et al., “The ContactSystem and Its Disorders,” in Blood: Principles and Practice ofHematology, Handin, R. et al., eds., J B Lippincott Co., Philadelphia,1995). If all of the prekallikrein were activated, then at least 500 nMof PEP-1 would be required for a stoichiometric inhibition ofkallikrein. An individual having 5 liters of plasma would thereforerequire about 18 mg of PEP-1 to achieve a plasma concentration of 500nM.

Another factor to consider is the threshold concentration of kallikreinrequired to induce a SIR in a patient. If the concentration of activekallikrein must be maintained below, e.g., 1 nM, then owing to its highaffinity for kallikrein, PEP-1 offers a significant advantage overaprotinin in the amount of protein that would be required to inhibitSIR. In particular, a concentration of PEP-1 of 1 nM would inhibit 99.6%of kallikrein present at 1 nM (i.e., only 0.4 pM free kallikreinremaining in the blood), whereas, an aprotinin concentration of 1 nMwould only inhibit 24.5% of the kallikrein present at 1 nM. Foraprotinin to inhibit 99% of the kallikrein at 1 nM, an aprotininconcentration in the plasma of at least 3 uM is required (i.e., 3000times higher concentration than for PEP-1).

For a patient undergoing CPB, an initial clinical dose of PEP-1 can beestimated from a recommended dose regimen of aprotinin (1×10⁶ KIU)mentioned above. Aprotinin is reported in a package insert to have asspecific inhibitory activity of 7143 KIU/mg determined using a dog bloodpressure assay. Therefore, 1×10⁶ KIU of aprotinin is equivalent to 140mg of aprotinin (i.e., 1×10⁶ KIU/7143 KIU/mg=140 mg of aprotinin). In apatient having a blood plasma volume of 5 liters, 140 mg corresponds toapproximately 4.3 TM aprotinin (molecular weight of aprotinin is 6512Daltons). The specific activity of aprotinin in the standard inhibitoryassay used for PEP-1 is 0.4 KIU/mg of polypeptide. A dose of 140 mgwould correspond to a loading dose for aprotinin of 56 KIU (140 mg×0.4KIU/mg=56 KIU). In contrast, since the specific activity of the PEP-1 KIpolypeptide is 10 KIU/mg in the standard inhibition assay, a dose ofonly 5.6 mg of PEP-1 would be required to provide the number of KIUsequivalent to 140 mg of aprotinin. In a patient with a plasma volume of5 liters, this corresponds to about 160 nM PEP-1 (molecular weight ofPEP-1 is 7054 Daltons), although a higher dose of the PEP-1 KIpolypeptide can be required if all of the plasma kallikrein (500 nM) isactivated and/or if this KI polypeptide is poorly distributed in apatient.

In some embodiment, the Kunitz domain polypeptide or KI polypeptide isadministered in a dose of about 1-500 mg/m², preferably about 1-250mg/m², 1-100 mg/m². For example, a KI polypeptide, e.g., a KIpolypeptide described herein, can be administered to a subject at riskfor cerebral ischemia, suffering from cerebrial ischemia, or who hassuffered a cerebral ischemic attack at a dose of 1-100 mg/m².

Furthermore, the KI polypeptides can be non-naturally occurring, andthey can be produced synthetically or recombinantly, as noted above,thereby avoiding potential contamination of transmissible diseases thatcan arise during isolation of a protein from a natural animal source,such as in the case of aprotinin, which is isolated from bovine lung.Increasingly important to administrative and public acceptance of atreatment or pharmaceutical composition comprising a polypeptide is theavoidance of possible contamination with and transmission to humanpatients of various pathological agents. Of particular interest for thesafety of proteins isolated from a bovine tissue is the elimination ofthe possible risk of exposure to viral mediated diseases, bacterialmediated diseases, and, especially, transmissible bovine spongiformencephalopathies.

As variants of the Kunitz domain 1 of the human LACI protein, fewer sideeffects are expected from administering the KI polypeptides to patientsthan for aprotinin, which is a bovine protein that is documented tocause anaphylactic and anaphylactoid responses in patients, especiallyin repeat administrations, such as second time CABG procedures.Additionally, the highly specific binding of the KI polypeptidesdescribed herein to kallikrein will effectively limit or eliminate thethrombotic tendencies observed with aprotinin, and reduce the problemsobserved with graft patency following CABG procedures.

The invention will be further described with reference to the followingnon-limiting examples. The teachings of all the patents, patentapplications and all other publications and websites cited herein areincorporated by reference in their entirety.

EXAMPLES Example 1 An Exemplary Kallikrein Inhibitor Polypeptide

An exemplary non-naturally occurring, KI polypeptide (PEP-1) wasidentified as a kallikrein binding polypeptide displayed on arecombinant phage from a phage display library. PEP-1 has the amino acidsequence:

(SEQ ID NO:2) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp

The molecular weight of PEP-1 is 7,054 Daltons.

The nucleotide sequence (SEQ ID NO:3) of the recombinant phage DNAencoding the PEP-1 amino acid sequence (amino acids 1-60 of SEQ ID NO:2) was isolated and sequenced by standard methods determined from therecombinant phage DNA.

PEP-1 was produced in amounts useful for further characterization as arecombinant protein in His4⁻ phenotype host cells of yeast strain Pichiapastoris.

Example 2 Construction of a Recombinant Plasmid to Express KIPolypeptides

The initial plasmid, pHIL-D2, was ampicillin resistant and contained awild type

His 4 of P. pastoris. The final DNA sequence comprising the codingsequence for the matα Prepro-PEP-1 fusion protein in the recombinantexpression plasmid pPIC-K503 is shown in FIG. 2. The DNA sequence ofpHIL-D2 was modified to produce pPIC-K503, as follows:

1. The BstBI site in the 3′ AOX1 region of pHIL-D2, located downstreamof the His 4 gene, was removed by partial restriction digestion,fill-in, and ligation, altering the sequence from TTCGAA (SEQ ID NO:23)to TTCGCGAA (SEQ ID NO:24). This modification was made in order tofacilitate and direct the cloning of the expression cassette into theplasmid.

2. The AatII site bear the bla gene located downstream of His4 wasremoved by restriction digestion, fill-in, and ligation modifying thesequence from GACGTC (SEQ ID NO:25) to GACGTACGTC (SEQ ID NO:26). Thismodification was made to facilitate the cloning of expression cassetteshaving AatII sites into the plasmid.

The DNA encoding PEP-1 was synthesized based on the nucleotide sequencefrom the original, kallikrein binding, display phage and consisted of450 base pairs (bp). The final DNA sequence of the insert in the pHIL-D2plasmid would be flanked by a 5′ AOX1 sequence and a 3′ AOX1 sequence(portions of which are shown in FIG. 2) and encode a fusion proteincomprising the matα Prepro signal peptide of S. cerevisiae fused to thestructural coding sequence for the PEP-1 KI polypeptide. The signalpeptide was added to facilitate the secretion of PEP-1 from the yeasthost cells. The oligonucleotides to form the insert were synthesized andobtained commercially (Genesis Labs, The Woodlands, Tex.) and linked bypolymerase chain reaction (PCR). The linked synthetic DNA encoding thematα Prepro-PEP-1 fusion protein was then incorporated by ligation intothe modified pHIL-D2 plasmid between the BstBI and EcoRI sites.

The ligation products were used to transform Escherichia coli strain XL1Blue. A PCR assay was used to screen E. coli transformants for thedesired plasmid construct. DNA from cell extracts was amplified by PCRusing primers containing the 5′AOX1 and 3′AOX1 sequences (see above andFIG. 2). PCR products of the correct number of base pairs weresequenced. Approximately 20-50 bp on either side of the cloning sites,in addition to the insert, were sequenced, and the expected sequence wasobtained. The final DNA sequence of the insert in the pHIL-D2 plasmid(to yield plasmid pPIC-K503) is shown in FIG. 2 along with portions offlanking 5′ and 3′ AOX1 sequences (SEQ ID NO:27) and corresponding aminoacid sequence of the fusion protein comprising the matα Prepro signalpeptide of S. cerevisiae fused to the structural coding sequence for thePEP-1 KI polypeptide (SEQ ID NO:28). A transformant with the desiredexpression plasmid construct, plasmid pPIC-K503, was selected forpreparing yeast cell lines for routine production of PEP-1.

Example 3 Manufacture of PEP-1 from Recombinant Yeast Cell Line

Spheroplasts of P. pastoris GS115 having the His4⁻ phenotype weretransformed with the expression plasmid pPIC-K503 (above) followinglinearization of the plasmid at the SacI site and homologousrecombination of the plasmid DNA into the host 5′AOX1 locus. Thephenotype of the production strain is His4⁺. The entire plasmid wasinserted into the 5′AOX1 genomic sequence of the yeast.

Isolates from the transformation were screened for growth in the absenceof exogenous histidine with methanol as the sole carbon source. Greaterthan 95% of the transformants retained the wild type ability to growwith methanol as the sole carbon source, demonstrating that the plasmidhad been inserted into the host genome by homologous recombinationrather than transplacement, and did not require exogenous histidine forgrowth, demonstrating that the plasmid was integrated into the hostgenome. Selected colonies were cloned. Small culture expression studieswere performed to identify clones secreting the highest levels of activePEP-1 into the culture medium. PEP-1 secretion levels in clarifiedculture supernatant solutions were quantified for PEP-1 levels by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andevaluated for kallikrein inhibition. A yeast clone was selected forPEP-1 production based on its high level of PEP-1 expression amongcultures sampled.

Master and working cell banks of PEP-1 producing P. pastoris wereprepared commercially, MDS Pharma Services, Bothell, Wash.). A standardproduction of PEP-1 in yeast comprised three steps: (1) preparation ofthe seed culture, (2) fermentation, and (3) recovery of the culture.

The seed culture step consisted of the inoculation of six flasks (300ml) containing sterile inoculum broth (yeast nitrogen base, potassiumphosphate, and glycerol, pH 5) with the contents of a single vial of aworking cell bank of PEP-1 producing P. pastoris. Flasks were inoculatedin an orbital shaker (300 rpm) for approximately 13 hours at 30° C.±2°C.

Fermentations were performed in a closed 100 liter Braun fermenterfilled with sterile broth. Each fermentation was initiated with thetransfer of the contents of the six seed culture flasks to thefermenter. After approximately 24 hours, the glycerol in the fermenterbecame exhausted and additional glycerol was added for approximately 8additional hours.

A mixed feed phase, which lasted approximately 83 hours, was theninitiated by the addition of a glycerol and methanol feed. At the end ofthis time, the fermentation was terminated, and the fermenter contentswere diluted with purified water.

The purification and processing of PEP-1 consisted of five steps: (1)expanded bed chromatography, (2) cation exchange chromatography, (3)hydrophobic interaction chromatography (HIC), (4) ultrafiltration anddiafiltration, and (5) final filtration and packaging.

The initial purification step consisted of expanded bed chromatography.The diluted fermenter culture was applied to the equilibrated columnpacked with Streamline SP resin (Amersham Pharmacia Streamline 200chromatography column, Amersham Pharmacia, Piscataway, N.J.). Thediluted fermenter culture was applied to the equilibrated column. Thecolumn was then washed (50 mM acetic acid, pH 3.0-3.5) in an up-flowmode to flush the yeast cells from the expanded bed. The top adaptor wasraised above the expanded bed enhance washing. The flow was stopped andthe bed was allowed to settle. The adaptor was moved down so that it wasslightly above the settled bed. The direction of the flow was reversed.The effluent was collected. Washing was continued in a downward modeusing 50 mM sodium acetate, pH 4.0. The effluent was collected. PEP-1was eluted from the column using 50 mM sodium acetate, pH 6.0. Theeluate was collected in a 50 liter container. The eluate was thenfiltered through a 0.22μ filter into a clean container located in thepurification site. Additional samples were collected for thedetermination of PEP-1 concentration.

A cation exchange chromatography step was then performed using thefiltered eluate from the expanded bed column. PEP-1 eluted from thecolumn using 15 mM trisodium citrate, pH 6.2.

Additional proteins were removed from the PEP-1 preparation byhydrophobic interaction chromatography (HIC). Prior to HIC, the eluatefrom the cation exchange column was diluted with ammonium sulfate. Theeluate was applied to the column, and the PEP-1 was eluted usingammonium sulfate (0.572 M) in potassium phosphate (100 mM), pH 7.0. Theeluate was collected in fractions based on A₂₈₀ values. All fractionswere collected into sterile, pre-weighed PETG bottles.

Selected fractions were pooled into a clean container. The pool wasconcentrated by ultrafiltration. The concentrated PEP-1 preparation wasimmediately diafiltered against ten volumes of PBS, pH 7.0.

A final filtration step was performed prior to packaging in order tominimize the bioburden in the bulk PEP-1. The bulk solution was filteredthrough a 22μ filter and collected into a sterile, pre-weighed PETGbottle. A sample was removed for lot release testing. The remainder ofthe bulk was dispensed aseptically into sterile PETG bottles and storedat −20° C.

Example 4 Kallikrein Inhibition Assay

The potency assay of KI polypeptides, such as PEP-1, described hereinwas a kinetic test, which measured fluorescence generation following thekallikrein-mediated cleavage of a substrate, prolylphenylalanylarginylamino methyl coumarin. A known amount of kallikrein was incubated withbuffer, with a serially diluted KI polypeptide reference standard, orserially diluted KI polypeptide test samples. Each sample was run intriplicate. The substrate solution was added, and the plate readimmediately using an excitation wavelength of 360 nm and an emissionwavelength of 460 nm. At least two each of the reference standard andsample curves were required to have an R-squared value of ≧0.95 to beconsidered valid.

Example 5 Neuroprotective Effect of Kallikrein Inhibitor Polypeptide atVarious Doses on Brain Ischemia/Reperfusion Injury

The presence of tissue kallikrein in the brain and the identification ofbradykinin B2 receptors on brain cells suggest that kinin system couldplay a role in the nervous system pathophysiology. Recent data showingthe effectiveness of bradykinin B2 receptor antagonists in reducingischemic brain damage further supports such a hypothesis.

In this example, a kallikrein inhibitor polypeptide (DX-88) is effectivein reducing neurological deficits and brain injury after transient focalbrain ischemia.

In vitro analysis was performed to determine whether DX-88 was able tocross the blood-brain barrier. Non-ischemic mice were treated iv withsaline or DX-88 (10 μg/mouse or 30 μg/mouse) and plasma was drawn 30 or60 min after administration. Thirty minutes after DX-88 treatment,plasma kallikrein inhibitory activity was 2 and 4 times higher in micetreated with 30 and 10 μg, respectively. Such a difference was not seenin plasma drawn 60 min after the infusion. In cerebral spinal fluid(CSF) from mice treated with 30 μg of DX-88, there was marked inhibitoryactivity with no difference between CSF drawn 30 or 60 min after DX-88administration.

Ischemia was induced by occlusion of the middle cerebral artery (MCAO).At the end of the ischemic period (30 min), the filament was removed andreperfusion allowed. Mice received different doses of DX-88 iv at thebeginning of the ischemic period. Twenty four hours after ischemia,neurological deficits and infarct size were evaluated. While salinetreated mice showed stable scores, those who received 30 mg of DX-88 hadsignificantly reduced general (13 and 10.5, median of saline and DX-88treated mice, respectively) and focal (26 and 15.5, median of saline andDX-88 treated mice, respectively) deficits scores. In these mice theischemic volume was also significantly reduced (22.86±5.82 mm³,23.04±4.34 mm³ respectively) compared to saline treated mice (63.12±7.69mm³). This study shows that: i) DX-88, in its active form, can crossrapidly the intact blood-brain barrier and thus reach brain tissue; ii)DX-88 has a significant neuroprotective effect against adverseconsequences of brain ischemia and reperfusion injury.

Example 6 Neuroprotective Effect of Kallikrein Inhibitor PolypeptideAdministered at Different Times During Brain Ischemia and ReperfusionInjury

Ischemia was induced by occlusion of the middle cerebral artery by MCAOusing a 6-0 monofilament. At the end of the 30 min ischemic period, thefilament was removed and reperfusion allowed. Mice received DX-88 iv atthe beginning of the ischemic period or at the end of it, duringreperfusion.

It was first analyzed whether DX-88 was able to cross the blood-brainbarrier and found that thirty minutes after treatment with 30 μg/mouseiv, a marked inhibitory activity was present in CSF. The same dose ofDX-88 was given at the beginning of ischemic period. Twenty four hoursafter ischemia, neurological deficits and infarct size were evaluated.While saline treated mice showed stable scores, those who received ofDX-88 had significantly reduced general (by 37.5%) and focal (by 50.0%)deficits scores. In these mice the ischemic volume was alsosignificantly reduced by 50.9%. When given at reperfusion, DX-88 wassimilarly effective in improving general (by 38%) and focal (by 50.1%)deficits scores as well as the ischemic volume that was reduced by 58%.

This study shows that: i) this specific kallikrein inhibitor can crossrapidly the intact blood-brain barrier and thus possibly reach braintissue in its active form; ii) it has a significant neuroprotectiveeffect against adverse consequences of brain ischemia and reperfusioninjury.

Other variations and embodiments of the invention described herein willnow be apparent to those of ordinary skill in the art without departingfrom the scope of the invention or the spirit of the claims below.

1.-13. (canceled)
 14. A method for preventing or reducing reperfusioninjury in a patient, the method comprising: administering to the patienta composition comprising an anti-plasma kallikrein antibody, in anamount effective to for preventing or reducing reperfusion injury in apatient.